16S Sequencing of Aedes aegypti reared at UTMB
Ae. aegypti Mosquitoes  Colonies of Ae. aegypti used in this study originated from Galveston (USA), Iquitos (Peru), Juchitan (Mexico), and Thailand. At UTMB, the generation of the colonies is not known but the colonies have been in the insectary since 2010 and reared continuously. The colonies were transferred to LSTM in 2018. At each institute the mosquito lines were housed under standard insectary conditions consisting of 28oC +/- 1 oC and 70% +/- 10% relative humidity with 12h:12h light dark cycle. Eggs were hatched in deionized water and larvae were fed fine ground tetramin fish food at both insectaries. Adults were held in Bugdorm cages at a density of 300-600 mosquitoes with constant access to 10% sucrose until being harvested. Mosquitoes were not bloodfed for this study. At LSTM colonies are fed fresh human blood from the NHS for egg production. At UTMB colonies are fed fresh defibrinated sheep blood Colorado Serum Company for egg production  DNA Extractions Three-five days post emergence, Ae. aegypti were cold anesthetized and females taken for surface sterilization. Individual mosquitoes were surface sterilized in ethanol 70% for 5 minutes followed by 3 washes in sterile PBS. DNA was extracted from 20 female mosquitoes from Galveston, Iquitos, Juchitan and Thailand using the QIAamp DNA Mini Kit (QIAGEN) following the manufacturer protocol with the following modifications: initial volume of 180ul of buffer ATL used for mosquito homogenization and final volume of 100ul nuclease-free water for DNA elution. No RNase A treatment was applied. For the UTMB samples, DNA from mosquitoes from Iquitos and Juchitan was extracted on 25 February 2021 and DNA from mosquitoes from Galveston and Thailand was extracted on 26 February 2021. No-mosquito controls were used for each extraction batch performed at UTMB and sequenced as negative controls.    Library Preparation and Sequencing Sequencing libraries for each isolate were generated using universal 16S rRNA V3-V4 region primers [22] in accordance with Illumina 16S rRNA metagenomic sequencing library protocols. DNA concentrations of each library were determined by Qubit and equal amounts of DNA from each barcoded library were pooled prior to sequencing. The samples were barcoded for multiplexing using Nextera XT Index Kit v2. The pooled libraries were diluted to 4 pM and paired-end 250 bp sequencing was performed on the Illumina Miseq using a MiSeq Reagent Kit v2 (500-cycles).